1. Field of the Invention
The field of the present invention concerns and relates to cryopreservation devices and methods.
2. Description of Related Art
As for the related art, organs which are harvested for transplant often need to be preserved in functional and transplantable condition for a period of hours or days to allow transport to the recipient.
Most common current practices for preserving harvested organs for transplant use ultra-profound hypothermia (between 0 and 5 degrees C.) combined with replacing the blood and/or submerging the organ in an organ preservation solution that consists of water, electrolytes, sugars, colloids, buffers, free radical scavengers, and metabolic precursors or metabolic suppressors. Sometimes, the organ preservation solution is continuously or intermittently perfused through the organ. This perfusion may or may not include the use of a heat exchanger and/or oxygenator to control temperature and to support metabolism. Sometimes different solutions are used to initially flush the organ, to store the organ without perfusion, or to perfuse the organ during storage. These methods generally do not allow for storage for more than three days, and most often (depending on the organ) only a few hours.
Previous efforts to extend this time to weeks or months have failed. Freezing organs, in which temperature is lowered below 0 degrees C., has failed to preserve transplantable organs because of the formation of ice. The experimental use of cryoprotective agents that reduce (but do not eliminate) ice formation are common in the storage of sperm, bacterial or cell cultures, or very small tissues. These agents, including organic solvents like glycerol or dimethyl sulfoxide, work on small sample sizes, but have not appreciably improved prospects for frozen organ storage.
Supercooled storage, where temperature is reduced slightly below 0 degrees C. and where ice formation has been suppressed by natural solutes, sugars, ice blockers, or anti-freeze proteins, has been similarly unsuccessful.
There has been some experimental success with vitrification of complex vascular organs. Vitrified organs or tissues are loaded with a high enough concentration of cryoprotectant agents that no ice forms even when cooled below the glass transition temperature of water-cryoprotectant mixtures (often below −120 degrees C.). Ice formation is prevented by replacing half or more of the water in the tissues with single or mixtures of organic solvents that penetrate cell membranes (such as dimethyl sulfoxide or glycerol), agents that do not penetrate the cell membrane (such as sugars, sugar alcohols, starches, proteins, and polymers), and agents that directly prevent the growth of ice from nucleating sites (ice blockers and anti-freeze proteins). This is generally accomplished in vascular organs by perfusing a gradually increasing concentration of cryoprotectant. Rabbit kidneys loaded with enough cryoprotectant to vitrify have consistently survived and provided sole support after re-implantation into experimental animals. At least one rabbit has survived indefinitely on a single kidney that had been vitrified and cooled to the glass transition temperature. This result has proven difficult to reproduce reliably.
Another experimental method for organ preservation has been persufflation. Persufflation is the process of flowing a gas through the vasculature of an organ, rather than blood or some other fluid. Persufflation that used oxygen to support metabolism has had some success, but provides results no better than conventional organ preservation solutions and hypothermia. Persufflation with hydrogen sulfide or carbon monoxide to suppress mitochondrial respiration is currently being experimented with. At least one attempt has been made to use persufflation to cool organs for frozen storage, but this proved unsuccessful due to the inherent problems of frozen storage.
The fundamental problem with persufflation is that the length of time an organ can be stored is similar to methods using organ preservation solutions and hypothermia. Vitrification below the glass transition temperature can, on the other hand, permit storage for years or decades.
Two problems are known to exist in vitrified organs and tissues. First, is the formation of thermo-mechanical fractures around and below the glass transition temperature. In vascularized tissue, organs and organisms of a size greater than a few cubic centimeters, thermo-mechanical fractures consistently transect blood vessels sufficiently to prevent successful recovery upon rewarming. Second, the cryoprotectant solutions that permit cooling without ice formation are themselves toxic and cause biochemical damage that makes viable recovery of the organ difficult.
Thermo-mechanical fractures are created by differential contractions in the tissues; and they may be caused by differences in coefficients of expansion in different tissue types, different cryoprotectant concentrations, by thermal gradients, and perhaps by other means. Masses of vitrifiable tissues larger than a few cubic centimeters consistently develop these large-scale fractures.
The tendency of tissue to fracture increases proportionally with the volume or domain size of the tissue. Suggestions for storage at just below the glass transition temperature may reduce—but in practice do not eliminate—fractures. Temperatures very close to the glass transition may also permit the growth of ice nucleation points that can make viable recovery of the organ more difficult by increasing the likelihood of ice forming during both cooling and rewarming. Furthermore, temperatures at or near the glass transition point (often below −120 degrees C.) may be insufficient for long-term banking of vascularized tissue, organs or organisms.
Cryoprotectant toxicity includes but is not limited to dehydration, membrane damage, destabilizing and denaturing proteins, oxidative damage, and metabolic disruption. The extent of this damage is roughly proportional to the exposure time of biological tissue to cryoprotectant at a given temperature. Generally, the longer the exposure time and the higher the temperature, the greater the toxic damage. Conversely, cooling rapidly reduces both exposure time and temperature, reducing toxicity.
Additionally, with vitrifiable organs, there exist critical cooling rates below which ice forms, and above which ice does not form. Likewise, there are critical warming rates below which ice forms on warming and above which ice does not form. These critical cooling and warming rates depend on the concentration of cryoprotectant or vitrification solution. Therefore, increasing both cooling and warming rates can reduce cryoprotectant concentration, effectively reducing cryoprotectant toxicity. Since cryoprotectant toxicity increases non-linearly with concentration, even small reductions in concentration can yield large decreases in toxicity.